| Atherosclerosis（AS）is a chronic inflammatory disease of arteries triggered by vascular endothelial cell injury.Its clinical manifestations are chronic inflammatory reactions in the vascular wall,forming atherosclerotic plaques in the intima of aorta,carotid artery or coronary artery.It is the pathological basis of ischemic heart disease,ischemic stroke,peripheral artery and other cardiovascular and cerebrovascular diseases.The disease is delayed by lipid-lowering,anti-inflammatory and antioxidant drugs,combined with stent and bypass and other interventional treatments,resulting in a huge economic burden on patients,the pathogenesis of the disease has not been fully clarified.Therefore,it is of great significance to explore the pathogenesis of AS for the treatment of cardiovascular diseases.It has been shown that vascular endothelial cell injury,monocyte infiltration and phenotypic transformation of vascular smooth muscle cells lead to the formation of arterial plaque.Among them,phenotypic conversion of VSMCs plays an important role.Under normal circumstances,transcriptional activators Myocardin bind with serum response factors（SRF）to regulate the expression of VSMCs contractile genes such as Sm22αandα-Actin maintain the contractile phenotype of VSMCs.However,the contraction gene expression of VSMCs is decreased under the stimulation of various factors after endothelial cell damage,and the transformation from contraction phenotype to synthetic phenotype is an important cellular event in the development of AS.Previous studies in our laboratory found that serum Response factor binding protein 1（SRFBP1）was a cofactor of Myocardin,and cooperated with Myocardin to activate the transcription of contractile marker genes of VSMCs cultured in vitro,thus inhibiting the proliferation of VSMCs and maintaining the contractile phenotype.Based on the previous experimental basis,this project uses loss-of-function strategy to explore the function of this gene in VSMCs in vivo.However,embryo death occurred during the construction of Srfbp1 knockout mice,and it was found by mouse embryo genotype identification that,it was found that wild:heterozygous:homozygous ratio of mouse genotypes before E9.5 was 1:2:1.However,the homozygote ratio decreased to 0 at E10.5.At the same time,E7.5-E9.5 embryos were dissected and no obvious abnormality was found in E7.5 and E8.5embryos,However,some embryosomes of E9.5 embryos showed atrophy.In order to determine the time of SRFBP1 function during embryonic development,E6.5-E14.5 mouse embryos were used to detect the change of SRFBP1 expression level.The results showed that SRFBP1expression started from E7.5 and continued to E14.5.Semi-thin sections of E9.5 embryos stained with toluidine blue showed pyknosis of the lethal embryonic cells and an abnormal site of heart formation,it is speculated that the embryonic death of Srfbp1-/-mice may be caused by cardiac abnormality.To investigate the function of SRFBP1 in VSMCs in vivo,Our analysis showed that the expression of Srfbp1 protein in the aorta of knocked out heterozygous(Srfbp1+/-)mice was decreased about 60%.Based on this,this study analyzed the effect of SRFBP1 on atherosclerosis by hybridizing Srfbp1+/-with Apolipoprotein E deletion(Apolipoprotein E dificient,Apoe-/-)of AS classical model mice.SRFBP1+/-mice were crossbred with Apoe-/-mice to obtain Srfbp1+/-/Apoe-/-mice,and bred with Srfbp1+/-/Apoe-/-and Srfbp1+/+/Apoe-/-mice for subsequent experiments.The mice were fed a high-fat and high-cholesterol diet for 5 months after the normal diet to the age of 1 month,After the sampling,the blood lipid level,the expression of Srfbp1 and VSMCs contractile genes（Sm22αandα-Actin）in different mouse artery tissues were analyzed.The results showed that the low expression of SRFBP1 did not affect the changes of blood lipid levels,the m RNA and protein levels of Srfbp1 and Sm22αandα-Actin genes related to the contraction of VSMCs decreased with the increase of age,suggesting that SRFBP1 was involved in the development of AS and was independent of the changes of blood lipid levels.Oil red O staining was used to analyze the lesion area of AS plaque in frozen sections of full-length aorta and cardiac aortic root.The results showed that AS plaque area in full-length aorta of Srfbp1+/-/Apoe-/-mice increased significantly,and the plaque area increased by 47.46%and71.67%after 2 and 5 months of high-fat and high-cholesterol feeding.The results of aortic root section analysis were similar,indicating that the low expression of SRFBP1 promoted the development of AS.At the same time,the necrotic core of Srfbp1+/-/Apoe-/-plaques increased by about 44.7%,indicating that SRFBP1 inhibited the development of AS by reducing the area of necrotic core of plaques.Immunofluorescence in plaque was performed using monocyte macrophage marker protein F4/80 and VSMCs marker proteinα-SMA.The results showed that the positive area ofα-SMA in Srfbp1+/-/Apoe-/-mice aortic root lesions increased by about22.47%,and the positive area of F4/80 decreased by about 31.85%,suggesting that the proportion of VSMCs in arterial plaques was increased after the low expression of SRFBP1,suggesting that the inhibition of SRFBP1 on AS mainly depends on its effect on VSMCs.Because SRFBP1 expression was down-regulated during the occurrence of AS,we overexpressed SRFBP1 vector by adeno-associated virus with vascular smooth muscle specific expression element,and injected AAV9-Srfbp1,AAV9-CON,and PBS into AS model mice by tail vein at one time.The function of SRFBP1 in maintaining the contrsystolic phenotype of VSMCs was further investigated.The results showed that compared with the AAV9-CON and PBS groups,the constriction related genes of aortic VSMCs in the AAV9-Srfbp1 group were significantly increased,Aortic lesion area and plaque lipid deposition were reduced by 42.9%and 57.2%respectively,and independent of changes in lipid levels,it was further verified that SRFBP1 inhibits the occurrence of atherosclerosis by regulating the expression of contractile proteins in VSMCs.In conclusion,SRFBP1 may play an important role in heart development and participates in the occurrence of AS by participating in the regulation of the expression of VSMCs contractile gene in arterial tissues,it provides a theoretical basis for the study of the pathogenesis of AS and some data support for AAV gene therapy of AS. |
